In our previous studies, we have taken a proteomic approach to characterize the components of the KSR1 scaffold during dynamic signaling events. Through this work, we have found that KSR1 translocates from the cytosol to the plasma membrane upon Ras activation and coordinates the assembly of a large multiprotein complex that functions to regulate the intensity and duration of ERK cascade signaling. More specifically, we have identified a hydrophobic motif in the proline-rich sequence of MEK1/2 that mediates constitutive binding to the KSR1 scaffold and find that KSR1 forms a ternary complex with B-Raf and MEK in response to growth factor treatment that enhances B-Raf-mediated MEK activation. Strikingly, we have also found that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback sites. Phosphorylation of the feedback sites attenuates ERK cascade signaling by promoting the dissociation of the B-RAF/KSR1/MEK complex and causing the release of KSR1 from the plasma membrane. In addition, we have found that KSR expression levels can alter the effects of ATP-competitive Raf inhibitors on oncogenic Ras/ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling. Due to success of the proteomic approach in elucidating the function and regulation of the KSR scaffolds, we have expanded our use of these techniques to investigate the mammalian CNK scaffold family, comprised of the CNK1, CNK2 and CNK3 proteins. Not surprising given the similar domain structure of the CNK family members, this analysis identified several common CNK-interacting proteins; however, it also revealed key differences in the CNK complexes that suggest important functional diversity. In particular, we found that CNK1 interacts with members of the cytohesin family of Arf guanine nucleotide exchange factors and that the CNK1/cytohesin interaction is critical for the activation of the PI3K/AKT cascade downstream of insulin and IGF-1 receptors. The insulin pathway is vital for energy metabolism and growth, and its dysregulation is a major contributor to human disease. These findings provide new mechanistic insight regarding the regulation of this important pathway and define a role for CNK1 as a regulator of both cytohesin function and insulin/IGF-1 signaling. During this fiscal year, we have also characterized the major binding partners of the CNK2 scaffold and find that CNK2 interacts with critical regulators of the Rho family GTPases. Moreover, our studies have revealed that CNK2 play a key role in integrating Arf and Rho family signaling during neuronal morphogenesis. Consistent with these findings, loss-of-function mutations in CNK2 have recently been reported in cases of non-syndromic X-linked mental retardation. Interestingly, increased expression of CNK2 has been observed in certain cancer types, indicating that CNK2 may also function to regulate Arf and Rho family signaling during tumorigenesis. We have also initiated a new study during this budget year in which we have characterized the binding partners of another Ras pathway modulator Sur8/Shoc2. Previous studies have shown that Sur8/Shoc2 interacts with proteins known to be involved in Ras pathway signaling, such as M-Ras, K-Ras, protein phosphatase 1 catalytic subunit, and a germline mutation in Sur8/Shoc2 has been associated with certain developmental Rasopathies. By identifying new binding partners of Sur8/Shoc2, we hope to better understand its function in normal and disease-associated signaling.